Protoporphyrin IX in serum of high-grade glioma patients: A novel target for disease monitoring via liquid biopsy

High-grade gliomas (HGG) carry a dismal prognosis. Diagnosis comprises MRI followed by histopathological evaluation of tissue; no blood biomarker is available. Patients are subjected to serial MRIs and, if unclear, surgery for monitoring of tumor recurrence, which is laborious. MRI provides only limited diagnostic information regarding the differentiation of true tumor progression from therapy-associated side effects. 5-aminolevulinic acid (5-ALA) is routinely used for induction of protoporphyrin IX (PpIX) accumulation in malignant glioma tissue, enabling improved tumor visualization during fluorescence-guided resection (FGR). We investigated whether PpIX can also serve as a serum HGG marker to monitor relapse. Patients (HGG: n = 23 primary, pHGG; n = 5 recurrent, rHGG) undergoing FGR received 5-ALA following standard clinical procedure. The control group of eight healthy volunteers (HCTR) also received 5-ALA. Serum was collected before and repeatedly up to 72 h after drug administration. Significant PpIX accumulation in HGG was observed after 5-ALA administration (ANOVA: p = 0.005, post-hoc: HCTR vs. pHGG p = 0.029, HCTR vs. rHGG p = 0.006). Separation of HCTR from pHGG was possible when maximum serum PpIX levels were reached (CI95% of tMax). ROC analysis of serum PpIX within CI95% of tMax showed successful classification of HCTR and pHGG (AUCROC 0.943, CI95% 0.884–1.000, p < 0.001); the optimal cut-off for diagnosis was 1275 pmol PpIX/ml serum, reaching 87.0% accuracy, 90.5% positive predictive and 84.0% negative predictive value. Baseline PpIX level was similar in patient and control groups. Thus, 5-ALA is required for PpIX induction, which is safe at the standard clinical dosage. PpIX is a new target for liquid biopsy in glioma. More extensive clinical studies are required to characterize its full potential.


Clinical procedure and sample collection
All experiments were performed in accordance with the declaration of Helsinki and with approval of the local ethics committee of the University of Münster and the Ärztekammer Westfalen-Lippe (2017-169-f-S); all patients gave informed written consent.Patients undergoing FGR with lesions suspicious for primary (n = 23, pHGG) or recurrent (n = 5, rHGG) HGG were included in the study (for details, see Table 1).Blood was collected before, during, and after surgery.
Patients received a standard oral dose of 20 mg/kg body weight 5-ALA (Gliolan®, medac, Wedel, Germany) four hours prior to induction of anesthesia.Treatment of patients followed the standard procedures in our clinic.All patients gave informed written consent.The standard 5-ALA-dose (5-aminolevulinic acid-HCl, pharmacy, University Hospital of Münster) was also administered to a healthy control group (HCTR, n = 8).A second healthy control group without 5-ALA administration (HCTRw, n = 11) was additionally included for baseline PpIX analysis (Table 1).
Data for tumor volume [cm 3 ], residual tumor volume after FGR [cm 3 ], body mass index (BMI), Eastern Cooperative Oncology (ECOG) performance status, classification according to the American Society of Anesthesiologists (ASA), medication and duration of anesthesia were collected, as well as clinical blood parameters (erythrocytes, leukocytes, hemoglobin, hematocrit, thrombocytes, total bilirubin, alanine-aminotransferase (ALT), aspartate-aminotransferase (AST), gamma-glutamyltransferase (gamma-GT), quick value (determined as international normalized ratio, INR), creatinine, urea) were evaluated in the context of the study.Tumor volume

Protoporphyrin IX quantification in serum
In blood samples with low expected PpIX levels (HCTRw; HCTR, pHGG, rHGG: pALA and ≥ 48 h aALA), 500 µl serum was prepared for liquid chromatography (LC)-MS analysis; otherwise (HCTR, pHGG, rHGG: 0.5-12.0h aALA), 100 µl serum was sufficient.The internal standard mesoporphyrin IX (MpIX, Merck KgaA, Darmstadt, Germany) was added to each serum sample, yielding 0.5 pmol/µl in the final extract for analysis.Liquid-liquid extraction (LLE) of PpIX from serum was achieved by adding four parts of water and ten parts of acetonitrile (ACN) to the serum volume in two steps.Samples were shaken for one hour after adding water for hemolysis, then ACN was added, and samples were shaken again for one hour for protein precipitation and porphyrin extraction.After LLE, samples were centrifuged (30 min, 16000 rcf), and the supernatant was transferred to an anionic-exchange solid phase extraction (ae-SPE) cartridge (Oasis MAX 3cc, Waters, Eschborn, Germany).The cartridge was washed with 2 ml 5% (v/v) ammonium hydroxide solution and 2 ml methanol.Porphyrins were eluted with 2 ml ACN containing 2% formic acid.The eluate was dried using a SpeedVac concentrator (2 h, 35 °C, Savant SPD 111V with vapor trap Savant RVT 5105, Thermo Fisher Scientific, Schwerte, Germany).The residue was reconstituted in 35 µl dimethyl sulfoxide (DMSO) for samples with low expected PpIX levels (HCTRw; HCTR, pHGG, rHGG: pALA and ≥ 48 h aALA) or in 40 µl DMSO for samples taken 0.5-12.0h aALA.Particles were removed prior to LC with centrifugal filter units (11 min, 12000 rcf, wwPTFE, pore size 2 µm, PALL Nanosep®, Dreieich, Germany).The workflow for blood sample collection and LC-MS analysis is summarized in Fig. 1.

Statistics
Statistical analysis was performed using SPSS software (Version 27, IBM, Germany).Data distribution was evaluated using histograms and Shapiro-Wilk test.If variables were not normally distributed, non-parametric procedures (Kruskal-Wallis with post-hoc Dunn-Bonferroni, Mann-Whitney U (MWU), Friedmann, Wilcoxon test) were used for comparison.One-way analysis of variance (ANOVA) with post-hoc Tukey test and t-tests were used if the criteria for these parametric tests were met.Spearmann-Rho correlation was additionally used for evaluation.All reported p-values were two-tailed.A p-value < 0.05 was considered statistically significant.
The area under the curve of PpIX serum levels (AUC SumPpIX ) was calculated according to the linear method (Eq. 1) and summed up for samples taken pALA until 12 h aALA.To evaluate the potential of serum PpIX to discriminate between healthy and pHGG patients, receiver operating characteristic (ROC) analysis was performed.

Baseline PpIX serum levels
Endogenous PpIX levels pALA and without drug usage were low (14 ± 11, 2-78 pmol/ml (mean ± standard deviation (sd), range), CI 95% 10-17 pmol/ml, n = 47), and there were no significant differences among the groups (Fig. 2, black square).There is no reference range for PpIX in serum for healthy individuals, but 2-15 pmol PpIX/ ml in plasma is considered normal for assessing porphyria based on HPLC-FLD analysis 34 .However, despite laborious protocols, PpIX often could not be reliably measured in the past 34 or was not detected in plasma samples at all 35 .PpIX plasma concentrations reported in the literature ranged from below the method-dependent limits of quantification to hundreds of nmol/l 31,35 .Reported results were strongly dependent on the analysis method 36 .
In contrast to RBC extract, no PpIX-suspicious signal at ~630 nm was apparent in plasma 29,30 , but different porphyrins were present in both matrices.Clearly, sensitivity and specificity of fluorophotometry for PpIX detection in plasma are insufficient, leading to questions concerning the reports about elevated PpIX levels in blood of cancer patients, which were determined with this method.LC coupled to MS has been used before to characterize coproporphyrin and PpIX in plasma from colorectal adenocarcinoma patients 25 and is an excellent means for the specific and reliable measurement and quantification of PpIX in serum.
We did not detect increased serum PpIX concentrations in HGG patients pALA by using LC-MS.Thus, to exploit the biomarker potential of PpIX in HGG, the stimulation of heme biosynthesis pathway with exogenous (1) www.nature.com/scientificreports/5-ALA was necessary.This is in accordance with previous findings in bladder 31,41 , liver 42 , colorectal 43 , and pancreatic 44 cancer, showing that the urinary porphyrin profile following 5-ALA administration could be a potential tumor biomarker.Differences in urinary porphyrin profiles pALA were not seen [42][43][44] .Additionally, in HGG, PpIX-containing extracellular vesicles were found in plasma, but only aALA and not pALA 32 .For our study, serum was analyzed because it is more native than plasma, contains fewer proteins, and porphyrin content and pattern are similar to plasma.Serum is closer to the in vivo conditions than plasma because no anticoagulant or other supplement is required after blood collection.Whole blood contains zinc PpIX (ZnPpIX) 45 , which is rapidly hydrolyzed to free PpIX and Zn 2+ under acidic conditions 45 .Consequently, a sum parameter of free and ZnPpIX would then be measured 36,46,47 .Moreover, ZnPpIX and free PpIX fluorescence overlaps 45,48 , which hampers reliable measurement of free PpIX next to ZnPpIX when using FLD.Serum, when properly collected and prepared, should be free of ZnPpIX.

Serum PpIX after 5-ALA administration
Due to the human body's capability for heme biosynthesis, increased PpIX formation was expected aALA in all groups.5-ALA is a small (131 Da) polar molecule and rapidly absorbed after oral administration.5-ALA plasma levels peak at 0.9 h, and the substance is quickly removed from the body with a terminal half-life of about three hours 35,49 .PpIX levels in blood also rapidly increase aALA 35,[49][50][51] .5-ALA supplementation seems to be well tolerated by the body 13 .Apart from the known light sensitivity of the skin 24 h aALA and rare elevation of liver enzymes, oral 5-ALA supplementation (20 mg/kg body weight) and the resulting induction of heme biosynthesis have no harmful side effects 13,52,53 .
In our study, the PpIX serum concentration rapidly rose aALA in all three groups (pHGG, rHGG, HCTR): 0.5 h after administration, the PpIX level was about ten-fold higher compared to that pALA.The measured values changed similarly in these groups up to 4.5 h.After that, they started to differ considerably (Fig. 3).
In pHGG, the PpIX level 48 h aALA was significantly ~2-fold higher than the baseline (Fig. 2, pHGG 48 h aALA vs. pALA: n = 19, Wilcoxon test: p = 0.008).In four pHGG patients, the blood sample was taken at 72 h aALA instead of 48 h aALA due to clinical procedures.In these four samples, PpIX had returned to the baseline.For HCTR and rHGG, there was no significant difference in the baseline PpIX level at 48 h aALA.In contrast, for pHGG, the 48 h level was still significantly higher than the baseline in all groups (MWU test: p < 0.001, Fig. 2), indicating more extended clearance in pHGG.
t Max was also different in HGG compared to HCTR; in both patient groups, PpIX levels peaked later (pHGG: 8.9 ± 2.3, 4.5-12.2h aALA; rHGG: 6.4 ± 1.6, 4.5-8.0h aALA, HCTR: 5.0 ± 1.6, 2.5-8.0 h aALA (mean ± sd, range)).PpIX formation and/or degradation was delayed in rHGG by 1.5 to almost 4.0 h in pHGG.Additionally, t Max showed high individual variance with ranges overlapping among the groups.The differences in t Max were significant for comparison between HCTR and pHGG (ANOVA: p < 0.001, post-hoc Tukey test: p < 0.001); rHGG showed a delay in t Max , but probably due to the small number of individuals (n = 5) this was not significant.The literature reports PpIX blood maxima within a range of 5-12 h aALA and large individual variations 50,54 , but mainly PpIX peaked within 7-9 h 49,54 .PpIX fluorescence in glioma tissue, as determined in the context of research www.nature.com/scientificreports/ in FGR, reached the highest values 7-8 h aALA for strong and 8-9 h for weak fluorescing samples 55 matching the values observed in the present study.Within 8-10 h aALA, PpIX tissue fluorescence decreased 55,56 .For skin, PpIX fluorescence maxima were measured between 6.5 h aALA at the back of the hand and 9.8 h at the forearm 51 .

Correlation with clinical parameters
PpIX results (PpIX Max , t Max , AUC SumPpIX ) were tested against tumor volume, residual tumor volume after FGR, BMI, ECOG and ASA score, duration of anesthesia [h], total of medications taken, and patient age using a nonparametric Spearmann-Rho approach.PpIX Max and AUC SumPpIX correlated significantly (r s = 0.918, p < 0.001, n = 28), which is reasonable because a generally higher formation of PpIX is related to a higher PpIX Max .Additionally, the duration of anesthesia and t Max correlated significantly (r s = 0.667, p = 0.003, n = 19).t Max occurred later with prolonged anesthesia, which slowed down 5-ALA/PpIX metabolism.It is well known that anesthetic agents affect global oxidative metabolism, cerebral blood flow, and endogenous regulatory mechanisms such as cerebral autoregulation, vasomotor reactivity, and neurovascular coupling 57 .We have also noted more extended clearance in pHGG patients.In healthy volunteers, serum PpIX returned to the baseline within 48 h aALA; at this time, it was still elevated in pHGG patients (Fig. 2).
No other correlations were found, especially no effect on PpIX Max and AUC SumPpIX was detected.There was also no effect of individual medication apart from an earlier t Max with the intake of proton pump inhibitor Pantoprazole (intake: t Max = 7.9 ± 2.3 h aALA, n = 22; no intake: t Max = 10.4 ± 1.5 h aALA, n = 6, t-Test: p = 0.020; pairwise comparison of medication groups using MWU or t-Test dependent on data distribution).t max was approximately equal in men and women, and PpIX Max was slightly increased in women.For both parameters, differences were not significant between the sexes.
A specific effect of the surgical procedure on PpIX formation or degradation was thus not evident.All evaluated blood parameters were within the reference range except for postoperatively lowered erythrocytes, hemoglobin, and hematocrit, probably due to the extensive volume administration during anesthesia causing blood dilution.These included liver parameters, which were particularly interesting because the liver plays a central role in heme biosynthesis and PpIX elimination 14,58 .It is assumed that PpIX is solely eliminated by hepatic clearance and secreted into the bile 14,[58][59][60] .The first step in this mechanism, the uptake of PpIX into hepatocytes, is thought to be a first-order reaction kinetic and thus only dependent on the PpIX concentration; PpIX uptake is slow compared to other organic anions like bilirubin 60 .Details on the mechanism of hepatic clearance remain unclear, but PpIX passes the liver unchanged 61 , and intact PpIX is detected in faeces 62 .The canicular secretion is considered as rate-limiting step 60 .
Applying the cut-off values for classification of rHGG patients (n = 5) and two additionally sampled patients (low-grade glioma (LGG) n = 1, radiation necrosis n = 1, Table 1), AUC SumPpIX yielded 5/7 (71%) correct classifications.Two rHGGs were falsely classified as harboring no tumor.PpIX within CI 95% of t Max classified 4/7 (57%) cases correctly; one rHGG was again incorrectly diagnosed as tumor-free.Two rHGG patients were inconclusive because PpIX levels within CI 95% of t Max scattered above and below the cut-off.The radiation necrosis and LGG patients were each classified correctly with both variables.As the two patients with unclear pathologies according to the preoperative MRI (LGG, radiation necrosis) were classified correctly, these results show the potential of PpIX to assist in glioma monitoring.Further data is required to establish PpIX as a blood biomarker and evaluate its diagnostic performance in larger cohorts.

Assessment of PpIX as HGG biomarker
Next to PpIX, other promising circulating biomarker candidates are CTCs, circulating tumor DNA (ctDNA), exosomes, micro RNA, and proteins 10 .While some of these candidates, e.g., CTCs and ctDNA, are highly specific, allowing even molecular tumor classification, they require elaborate isolation techniques and suffer from low sensitivity of tumor detection 10 .In the case of micro RNA and proteins, specificity is often lacking 10 ; the link between micro RNA or protein biomarker candidates and the glioma tumor is often missing.PpIX is an established optical tissue marker during HGG resection [11][12][13]  Additionally, serum PpIX determination is more robust than CTCs or ctDNA analysis and requires less laborious protocols.For instance, the LC-MS workflow, which was developed for this clinical proof-of-concept study, can easily be transferred to any laboratory with a mass spectrometer.Although discrimination of pHGG from HCTR via serum PpIX was successful in our study, questions requiring further elucidation studies remain.For instance, the impact of the BBB permeability is unclear.BBB disruption will probably affect the sensitivity of the PpIX biomarker concept.The exact mechanism of PpIX accumulation in HGG aALA is not entirely clear yet 13,15 .Still, BBB disruption is essential for the uptake of 5-ALA in malignant glioma 13,[19][20][21]63 .
Moreover, elevated serum PpIX may not exclusively be related to HGG aALA.Instead, porphyrins have been investigated in a variety of oncological diseases, including prostate tumors 28,64 , breast cancer 26 , renal cell carcinoma 27 , colorectal cancer 25,43 , bladder cancer 31,41 , hepatocellular carcinoma 42 and pancreatic cancer 44 .It was extrapolated that the accumulation of porphyrins is common to almost all types of cancer [26][27][28] and that the specific measurement of PpIX is advantageous for cancer screening in general 31 .Still, most studies lack evidence on whether porphyrins in general or a specific porphyrin, e.g., PpIX was elevated.Due to nonspecific analytical methodologies, porphyrin discrimination was often not achieved [25][26][27][28][29]65 . Morover, lead poisoning, iron deficiency anemia, and porphyria can cause elevation of blood porphyrins 24,66 .While serum PpIX alone may lack specificity for initial glioma diagnosis, it could assist in surgical decision-making.Additionally, PpIX has the potential to aid in monitoring glioma recurrence and could possibly replace the need for repeated MRI scans with contrast agents.It could be standard practice to perform an MRI scan only after PpIX serum analysis suggests tumor progression.Furthermore, serum PpIX may help distinguish between true glioma progression and pseudoprogression, which can be challenging to discriminate using solely imaging techniques (MRI, 18 FET-PET) 3 .Often, histopathological evaluation of biopsied tissue is required for clarification 3 .

Limitations of the conducted study
This first proof-of-concept study included only a small number of healthy controls and patients.Thus, no separation into a discovery and validation cohort for the potential biomarker was possible.Moreover, patients were anesthetized and treated according to standard clinical procedures, while healthy controls were not.We evaluated this potential bias by analyzing clinical parameters.No specific effect on heme biosynthesis and the total amount of synthesized PpIX (PpIX Max , AUC SumPpIX ) was detectable.Therefore, classification analysis was feasible, and ROC analysis yielded promising results.Since anesthesia affected the 5-ALA/PpIX metabolism by shifting PpIX maxima to later time points for potential diagnostic use, it will be necessary to determine the time kinetics of PpIX serum levels in patients unaffected by surgery.

Conclusion
Discrimination between HGG patients and healthy controls is feasible by serum PpIX analysis following oral 5-ALA administration.We detected ~200-fold maximum serum levels compared to endogenous levels in pHGG in anesthetized patients between 4.5 and 12.2 h aALA (CI 95% 7.9-9.9h aALA) undergoing FGR.This first study discovered the potential of PpIX for a novel liquid biopsy approach in HGG.A test on a single blood sample drawn within the proper timeframe could indicate tumor presence or recurrence.Ultimately, these results still need to be proven in further studies with larger cohorts and an adapted study design.

Figure 1 .
Figure 1.Workflow for clinical sample collection and subsequent LC-MS analysis, created with BioRender.com.

Figure 3 .
Figure 3.Time kinetic of PpIX formation in serum from 0.5 to 12.0 h aALA.Kruskal-Wallis with post-hoc Dunn-Bonferroni test was used to compare the groups at each time

Figure 4 .
Figure 4. ROC analysis of AUC SumPpIX and PpIX levels within CI 95% of t Max in HCTR and pHGG.Points indicate the optimal cut-off values closest to the upper left corner; the diagonal solid line is the reference line.

Table 2 .
Calculated characteristics of test performance for the variables AUC SumPpIX and PpIX CI 95% t Max for discrimination of HCTR from pHGG (positive predictive value (PPV), negative predictive value (NPV)).